doi: 10.1161/hq0801.094222
© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
In Vivo Anti-Inflammatory Effect of Statins Is Mediated by Nonsterol Mevalonate Products
From the Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, and the Department of Medicine and Vascular Pharmacology, Consorzio Mario Negri Sud, Chieti (M.B.D.), Italy.
Correspondence to Luisa Diomede, Istituto di Ricerche Farmacologiche, "Mario Negri," Via Eritrea 62, 20157 Milano, Italy. E-mail diomede{at}irfmn.mnegri.it
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Abstract |
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Abstract—— This study set out to clarify whether the inhibition of sterol or nonsterol derivatives arising from mevalonate biotransformation plays a major role in the in vivo anti-inflammatory action of statins. Hepatic synthesis of all these derivatives was inhibited in mice by administered statins, whereas squalestatin inhibited only sterol derivatives. Using a short-term treatment schedule, we found that statins reduced the hepatic activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase without affecting blood cholesterol. This treatment inhibited lipopolysaccharide- and carrageenan-induced pouch leukocyte recruitment and the exudate production of interleukin-6, monocyte chemotactic protein-1, and RANTES. Coadministration of mevalonate reversed the effect of statin on leukocyte recruitment. The inhibition of sterol synthesis by squalestatin did not have any anti-inflammatory effect, indicating that the biosynthesis of nonsterol compounds arising from mevalonate is crucial for the in vivo regulation of cytokine and chemokine production by statins. Their inhibition by statins may account for the reported anti-inflammatory effects of these drugs and may provide a biochemical basis for the recently reported effects of statins in the prevention of cardiovascular disease and mortality.
Key Words: statins • HMG-CoA reductase • squalene synthetase • interleukin-6 • chemokines
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Introduction |
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Lipid-lowering drugs offer one of the most effective therapeutic approaches used in clinical practice for the prevention and treatment of atherosclerosis.1 Statins, a well-known class of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are active in the primary and secondary prevention of coronary heart disease and are the drugs most widely used for these purposes.2–5 Recent trials showed that statins reduce the risk of cardiovascular events even in the absence of a significant drop of blood cholesterol levels,6,7 suggesting that the benefits of statin therapy may also be ascribed to their action on nonlipid factors involved in inflammation-fibroproliferation, an important feature of atherosclerosis.8–11 The expression of proinflammatory cytokines, growth factors, cell-adhesion molecules, and proteins increased in extracellular matrix during the remodeling of atherosclerotic lesions.12 Therefore, the anti-inflammatory properties of statins might be an additional positive factor in controlling atherosclerosis and postangioplasty restenosis.
See p 1256
Statins prevent the conversion of HMG-CoA to mevalonic acid, and hence also the synthesis of bioactive sterol and nonsterol metabolic intermediates deriving from the cholesterol synthesis pathway.13 Inhibition of HMG-CoA reductase activity in monocytes14 and rat mesangial cell lines15,16 treated with lipopolysaccharide (LPS), granulocyte-macrophage colony–stimulating factor (GM-CSF), and phorbol myristate acetate, reduced the production of interleukin (IL)-8, IL-6, and monocyte chemotactic protein-1 (MCP-1), all factors involved in plaque formation and stabilization.12 A reduction of glomerular macrophage influx in nephrotic rats after lovastatin treatment, due to reduced expression of MCP-1, was reported,17 suggesting that the inhibition of mevalonate synthesis may influence leukocyte recruitment at the infection site. We recently showed that mevalonate-derived products participate in the regulation of the response to inflammation, because in vivo, lovastatin reduced the ability of leukocytes to migrate at the infection site by inhibiting chemokine production.18–20
In the light of these observations, a non–lipid-mediated anti-inflammatory action of statins could not be ruled out and would appear to be relevant as a mechanism underlying the inflammatory-fibroproliferative phenomenon that accompanied the antiatherosclerotic process. We therefore designed experiments to clarify whether the inhibition of sterol or nonsterol derivatives originating from the biotransformation of mevalonate is important in controlling inflammation (Figure 1). We used lovastatin, pravastatin, and simvastatin as inhibitors of the synthesis of sterol and nonsterol intermediates13,21,22 and squalestatin as a selective inhibitor of the synthesis of sterol derivatives only23–25 (Figure 1). We evaluated the anti-inflammatory effect of these molecules using the in vivo air-pouch model of local inflammation26,27 and measuring leukocyte migration and chemotactic cytokine production.
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Methods |
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Reagents
Lovastatin and simvastatin were kind gifts from Merck Sharp and Dohme (Rahway, NJ) and were converted to the active compound as previously described.22 Pravastatin was obtained from Menarini (Italy) and dissolved in 0.9% NaCl. Squalestatin was kindly provided by Glaxo Wellcome (UK). Iota carrageenan, carboxymethylcellulose (CMC), mevalonolactone, mevalonolactone-5-[3H] (ethanol solution; specific activity 10 000 to 40 000 mCi/mmol), and fluorescent isothiocyanate–conjugated BSA (FITC-BSA) were from Sigma Chemical Co. Bacterial LPS (from Escherichia coli 055:B5) was from Difco. 3-Hydroxy-3-methyl[3-14C]glutaryl-coenzyme A (buffered aqueous solution; specific activity 50 to 62 mCi/mmol) and RS-[2-14C]mevalonolactone (toluene solution; specific activity 50 to 62 mCi/mmol) were from Amersham. Rhodamine G was from Merck. All other reagents were of analytical grade.
Animals
Male CD1 mice, 18 to 20 g body weight (Charles River, Como, Italy), were used. The animals were housed at constant temperature (20±1°C) and relative humidity (60±10%) and supplied ad libitum with water and a standard diet. Procedures involving animals and their care were conducted in conformity with national and international laws and policies (EEC Council Directive 86609, OJ L358, 1, 12 December 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana no. 10, 18 February 1992; Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996).
Air-Pouch Model
Subcutaneous dorsal pouches were created in mice as illustrated in Figure 2, by injection of 5 mL of air and by reinjection, 3 days later, of 3 mL of air.26,27 On day 6, 0.2 µg LPS per mouse in 1 mL of 0.5% CMC (Figure 2A) or 1 mL 1% iota carrageenan in sterile saline per mouse (Figure 2B) was injected into the pouches. Controls received the corresponding vehicle alone. The anti-inflammatory effect of 1 to 10 mg/kg lovastatin in 0.5% CMC, 10 mg/kg simvastatin in 0.5% CMC, or 10 mg/kg pravastatin in 0.5% CMC was investigated by oral administration of these drugs to pouch-bearing mice 20, 12, and 0.5 hours before LPS (Figure 2A) or 0.5 hour before and 8 and 20 hours after carrageenan (Figure 2B). The anti-inflammatory effect of 2 to 20 mg/kg squalestatin dissolved in saline was tested by injection of the drug subcutaneously to pouch-bearing mice 44, 20, and 0.5 hours before LPS (Figure 2A) or 24 and 0.5 hours before and 23 hours after carrageenan (Figure 2B). The effect of 4.5 mg/kg of indomethacin was determined by oral administration to pouch-bearing mice 0.5 hours before and 8 and 20 hours after carrageenan. We tested whether 10 mg/kg mevalonate in saline reversed the anti-inflammatory effect of 10 mg/kg lovastatin by injecting it intraperitoneally 20, 12, and 0.5 hours before LPS. Controls received the vehicles alone. At different times after LPS or carrageenan treatment (4 hours and 24 hours, respectively, Figure 2), the animals were anesthetized, blood was collected from the retro-orbital plexus, and then they were killed by cervical dislocation. The pouches were washed with 1 mL of saline, and the liver was excised. The lavage fluid was immediately cooled on ice, the volume was recorded, and 50 µL was used for the count of total leukocytes after staining with erythrosine B dye. The remaining exudate was centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatant was stored at -20°C until needed.
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Vascular Permeability
Pouch-bearing mice were given 10 mg/kg lovastatin orally 0.5 hour before and 8 and 20 hours after carrageenan (Figure 2B) to investigate the effect on vascular permeability.26 Immediately before the injection of 1 mL 1% iota carrageenan per mouse into the pouch, 0.6 mg FITC-BSA in 0.2 mL saline per mouse was injected into the tail vein. Thirty minutes after carrageenan, the animals were anesthetized, blood and exudate were collected, and serum and exudate fluorescence was measured at an excitation wavelength of 490 nm and excitation of 521 nm. Fluorescence in the pouch was calculated as percentage of serum to exudate ratio.
Cholesterol Assay
Serum total cholesterol was assayed by a standard enzymatic method (Sigma).
HMG-CoA Reductase Activity
The hepatic activity of HMG-CoA reductase was determined in statin- and squalestatin-treated mice as described by Kita et al.22 Briefly, liver homogenates (100 to 150 µg of protein) were resuspended in solution A (20 mmol/L imidazole, pH 7.4, 5 mmol/L dithiothreitol, and 5 U alkaline phosphatase) and incubated for 30 minutes at 37°C. Then 87 µmol/L of [14C]HMG-CoA in solution B (40 mmol/L D-glucose-6-phosphate, 12 mmol/L dithiothreitol, 4 mmol/L NADPH, and 10 mmol/L EGTA) was added, and samples were incubated for 60 minutes at 37°C. The reaction was stopped with 18.5% HCl, and after the addition of 29 µmol/L [3H]mevalonolactone as internal standard, samples were incubated for 18 hours at 4°C. After centrifugation at 3000 rpm for 20 minutes, [3H]mevalonolactone and [14C]mevalonolactone were copurified from each sample by elution on a Kiesegel plate (Merck) with acetone:benzene (1:1 vol/vol) as mobile phase and identified with rhodamine G. The Rf value of the band was assessed by an unlabeled mevalonolactone standard. The [3H]mevalonolactone and [14C]mevalonolactone radioactivities were counted with a ß-counter scintillator (Beckman), and the activity of HMG-CoA reductase was expressed as pmol [14C]mevalonolactone formed · min-1 · mg protein-1. Protein concentration was determined with a Bio-Rad protein assay (Bio-Rad Laboratories).
Cytokines and Chemokines
The levels of IL-6 were measured as hybridoma growth factor activity on the 7TD1 cell line.28 MCP-1 level was measured by a specific sandwich ELISA (Benfer-Scheller, Milan, Italy) with a sensitivity limit of 40 pg/mL. RANTES level was determined with a specific mouse ELISA (R&D Systems) with a sensitivity limit of 2 pg/mL.
Statistical Analysis
Data are expressed as the mean±SD. Student’s t test and Duncan’s test for multiple comparisons were used, because the data were conforming to the normality test (Kolmogorov-Smirnov test for deviations from Gaussian distribution, GraphPad Prism 2.0a for Power Macintosh, GraphPad Software Inc).
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Results |
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To discriminate the hypolipidemic effect of statins from their anti-inflammatory potential, we initially set out to identify a short-term treatment schedule that significantly lowered hepatic HMG-CoA reductase activity without affecting total blood cholesterol. We gave pouch-bearing mice 3 consecutive oral doses of lovastatin from 1 up to 10 mg/kg and evaluated the lipid profile. No changes were observed, although there was a dose-related reduction in the hepatic activity of HMG-CoA reductase (Figure 3A). Three consecutive doses of 1 mg/kg did not significantly inhibit the hepatic enzyme activity, but 5 mg/kg lovastatin caused a 47% reduction. The maximal effect was observed with 10 mg/kg of lovastatin, which reduced the enzyme activity by 56% (Figure 3A).
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The anti-inflammatory action of lovastatin was then evaluated by determination of its effect on the number of leukocytes recruited into the pouch 24 hours after 1% carrageenan or 4 hours after 0.2 µg LPS per mouse (Figure 2). Lovastatin 1 to 10 mg/kg in 0.5% CMC was administered orally to mice 0.5 hour before and 8 and 20 hours after carrageenan or 20, 12, and 0.5 hours before LPS. As shown in the Table, lovastatin affected leukocyte recruitment only at doses that significantly inhibited hepatic HMG-CoA reductase. Independently of the inflammatory stimulus applied, 1 mg/kg lovastatin did not affect the ability of leukocytes to migrate at the infection site, whereas the dose of 5 mg/kg reduced the number of total leukocytes recruited by carrageenan or LPS significantly, by 26% and 31%, respectively (Table). The maximal effect was observed at 10 mg/kg, with a 64% and 52% of inhibition after carrageenan or LPS, respectively (Table).
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The effect of lovastatin on leukocyte recruitment was not specific for polymorphonuclear cells or monocytes, because the percentage of these cells in the exudate was not affected by statin treatment (data not shown). Thus, lovastatin significantly reduced leukocyte migration at the site of inflammation, this effect being related to its inhibition of hepatic HMG-CoA reductase.
The anti-inflammatory power of lovastatin was compared with that of indomethacin, a known anti-inflammatory drug.26 Indomethacin at 4.5 mg/kg significantly (P<0.01 by Student’s t test, n=6) reduced the number of leukocytes recruited into the pouch by carrageenan, from 6.1±3.1x106 to 3.6±1.7x106 cells. A similar effect was obtained with 10 mg/kg of lovastatin (8.2±0.13x106 cells with carrageenan alone and 4.9±0.9x106 cells with lovastatin and carrageenan, P<0.01 by Student’s t test, n=6), indicating that the anti-inflammatory power of this statin is comparable to that of indomethacin.
The anti-inflammatory effect of two other statins, pravastatin and simvastatin, was also considered. In pouch-bearing mice, 3 doses of 10 mg/kg of either drug, administered according to the scheme illustrated for statins in Figure 2, significantly reduced (P<0.05, treated mice versus controls, Student’s t test, n=6) hepatic HMG-CoA reductase activity by 
30% (controls, 6.3±0.5 pmol · min-1 · mg protein-1; pravastatin-treated mice, 4.4±0.3 5 pmol · min-1 · mg protein-1; simvastatin-treated mice, 3.9±0.6 pmol · min-1 · mg protein-1). As shown in Figure 4A and 4B, independently of the inflammatory stimulus applied, 10 mg/kg pravastatin or simvastatin reduced the number of leukocytes recruited into the pouch significantly, to the same extent as lovastatin.
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We then designed experiments to determine whether the effect of lovastatin on leukocyte recruitment was specifically related to its ability to inhibit the synthesis of molecules arising from mevalonate. Pouch-bearing mice were given 10 mg/kg lovastatin PO and 10 mg/kg mevalonate IP 20, 12, and 0.5 hours before LPS. As shown in Figure 4C, mevalonate alone did not affect the number of leukocytes recruited into the pouch by LPS, whereas, when given with lovastatin, it completely reversed the inhibitory effect of statin. Thus, the anti-inflammatory effect of statins is due to their inhibition of mevalonate-derived isoprenoid synthesis.
The effect of blocking the activity of the enzyme that catalyzes the first step of the cholesterol pathway, after it branches to various nonsterol products, was then investigated. Three consecutive doses of squalestatin (2 to 20 mg/kg), a specific inhibitor of squalene synthetase, were administered to pouch-bearing mice according to the scheme illustrated in Figure 2, and the effect on the lipid profile was considered. A dose-related effect was observed (Figure 3B). Squalestatin at 2 mg/kg was ineffective, whereas 10 and 20 mg/kg lowered serum cholesterol significantly, by 30% and 48%, respectively. This reduction was accompanied by a compensatory increase in hepatic HMG-CoA reductase activity21 of 2-fold and 4-fold, respectively, at doses of 10 and 20 mg/kg (Figure 3B).
To investigate the effect of 2 to 20 mg/kg of squalestatin on leukocyte migration, we gave it to pouch-bearing mice 24 and 0.5 hours before and 23 hours after carrageenan or 44, 20, and 0.5 hours before LPS. Squalestatin did not affect the number of leukocytes recruited into the pouches by LPS (Table) or by carrageenan (data not shown). This indicates that this drug has no appreciable anti-inflammatory activity, because the inhibition of sterol synthesis alone was not enough to affect the leukocyte migration during the local inflammatory process.
To clarify whether the effect of the inhibition of sterol-derived and non–sterol-derived products on leukocyte extravasation was linked to cytokine production, we measured IL-6, a proinflammatory cytokine produced both by vascular endothelium and by leukocytes, in the pouch exudate.27 As shown in the Table, the inhibition of inflammation-induced leukocyte recruitment observed with lovastatin was paralleled by a dose-dependent reduction of IL-6 levels. Similar effects were obtained with 10 mg/kg of pravastatin or simvastatin (data not shown). Squalestatin, which did not reduce the number of leukocytes recruited into the pouch, did not affect IL-6 production (Table).
The effect of lovastatin on the exudate production of 2 CC chemokines, MCP-1 and RANTES, was also considered. As shown in Figure 5, the effect of lovastatin on chemokine production was linked to the inflammatory stimulus applied. When carrageenan was used, 10 mg/kg lovastatin significantly lowered only RANTES exudate levels, whereas in mice treated with LPS, only exudate MCP-1 production was significantly reduced.
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Finally, because changes in the permeability properties of endothelium may affect leukocyte extravasation,26,27 we investigated whether the effect of the statins could be partially attributed to a modification of the pouch vascular permeability. The fluorescence of pouch exudate, quantified by FITC-BSA, was the same as in serum, in control (3.53±0.6%) and lovastatin-treated (3.54±0.7%) mice, 30 minutes after carrageenan, indicating that changes in vascular permeability are not involved in the statins’ reduction of pouch leukocyte recruitment.
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Discussion |
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This study focused on the biochemical pathways underlying the anti-inflammatory effect of statins. Our findings were generated according to a protocol designed to study the anti-inflammatory effects of inhibitors of sterol and nonsterol products arising from mevalonate biotransformation. The short treatment schedule used, in the case of lovastatin, did not reduce the levels of circulating cholesterol but caused a significant drop of hepatic HMG-CoA reductase activity. In the same experimental conditions, squalestatin, unlike lovastatin, caused a rapid drop of circulating cholesterol levels, and through the negative feedback mechanism controlling cholesterol synthesis,13 it induced hepatic HMG-CoA reductase activity.
The ability of the statins and squalestatin to regulate the early events of local inflammation, particularly cell migration and cytokine production, was investigated in the air-pouch model26,27 applied to mice, because we were not concerned with the sensitivity or the resistance of the species to atherosclerosis. This approach takes into account the role of the vascular endothelium as a regulator of leukocyte extravasation from vessels into inflamed tissues.27 Pouch vascular cells produce large amounts of cytokines, particularly IL-6, which is the major modulator of acute-phase gene expression during inflammation and plays a positive role in the local inflammatory reaction by amplifying leukocyte accumulation.27,29 Moreover, IL-6 is produced during the inflammatory-fibroproliferative phenomenon that accompanied the atherosclerotic process by a variety of cell types, including monocytes, smooth muscle cells, and endothelial cells, all involved in the plaque synthesis and in promoting its instability.29–31 All these characteristics made the air-pouch model a suitable model for studying the effect of inhibitor of sterol and nonsterol products on the cellular and humoral components of the inflammatory process.
The different anti-inflammatory effects displayed by statins and squalestatin depended on their different pharmacological targets: HMG-CoA reductase and squalene synthetase.13,23 Only the inhibition of the former by the statins was accompanied by a reduction of the ability of leukocytes to migrate into the pouch, because of the inhibition of IL-6, MCP-1, and RANTES production. Inhibition of sterol synthesis by squalestatin did not have any anti-inflammatory effect despite a significant drop of circulating cholesterol and the consequent stimulation of HMG-CoA reductase activity. The inhibitory effect of lovastatin on leukocyte recruitment was reversed when mevalonate was given at the same time, indicating for the first time that the biosynthesis of nonsterol compounds arising from mevalonate is crucial for the in vivo regulation of inflammation-induced production of cytokines and chemokines.
It is difficult to define in vivo the specific molecular mechanism at the basis of the effect of statin on cell migration because of the complexity of the cholesterol biosynthesis (Figure 1). These data indicate that the effect of statin passes through the inhibition of HMG-CoA reductase activity, affecting the levels of mevalonate, the precursor of isoprenoids. These are required for important cellular functions, such as the assembly of glycoproteins, heme, and GTP-binding proteins and for the regulation of cell proliferation.13 Increasing evidence suggests that statins are able to downregulate IL-6 and MCP-1 transcription as a consequence of interference with the sGTP binding proteins/nuclear factor-
B (NF-B) transduction pathway.30,31 In fact, NF-B is the key factor that promotes the transcription of both these cytokines.32 In inflammatory conditions, NF-B is activated through sGTP binding proteins (Ras-Rho) that, in turn, require posttranslational modification involving nonsterol mevalonate-derived compounds to be active.30,31
Chemotaxis is also a vital step in the development of atherosclerosis and for restenosis, because it guides the migration of leukocytes into the artery wall.12,33 MCP-1 and RANTES appear to be involved in the pathogenesis of atherosclerosis.34,35 In particular, mice with genetic inactivation of MCP-1, or of its CC-chemokine receptor 2 (CCR2), are reported to be considerably resistant to the development of atherosclerosis.36,37 Therefore, the fact that lovastatin inhibits the production of these chemokines may represent an additional pharmacological mechanism important for the reduction of atherosclerotic lesions.
The difference observed in the downregulation of lovastatin on MCP-1 and RANTES depending on the inflammatory stimulus may be related to the kinetics of synthesis of these chemokines. In fact, MCP-1 was detected as early as 2 hours after challenge with an inflammatory stimulus (zymosan) in the peritoneal cavity, with a maximal rate between 2 and 4 hours.38 After this peak, MCP-1 levels returned almost to the basal value within 8 hours.38 On the contrary, the kinetics of RANTES synthesis and secretion is slower, because its production required at least 8 hours.39 As a consequence, in the LPS-stimulated mice, MCP-1 was probably the main chemokine involved in the inflammatory response, whereas in the carrageenan-stimulated group, the role of RANTES was more important.
In summary, our data demonstrate that in vivo, statins show a specific anti-inflammatory effect mediated by the inhibition of nonsterol mevalonate-derived compounds, whose deficiency downregulates IL-6, MCP-1, and RANTES production. Further studies are necessary to establish whether the ability of the statins to reduce the occlusive atherosclerotic lesions may be linked, in addition to their lipid-lowering effect, also to their anti-inflammatory action, and whether this effect may interfere with the very early stage of atherogenesis or throughout the pathological process.
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Acknowledgments |
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Diego Albani is a fellow of the Fondazione Angelo and Angela Valenti, and Paolo Fruscella is a fellow of the Alfredo Leonardi and Gustavus and Louise Pfeiffer Research Foundation.
Received February 11, 2001; accepted May 4, 2001.
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